Identification of the components of transcription complexes and the genes associated/regulated by them are at the heart of all modern discoveries. As key epigenetic players with essential biological functions, histone deacetylases (HDACs) are among the most frequently analyzed proteins by chromatin immunoprecipitation (ChIP). To this end, ChIP is a successful and widely used technique to identify regions of DNA interacting with the proteins of interest. However, current ChIP technologies have several limitations that frequently pose serious challenges. As an alternative to antibody-based ChIP assays, we propose to develop a novel technology where we will use our novel small molecule HDAC photoreactive probes (PRPs) to interrogate HDAC binding to chromatin. Among many advantages, this technology is capable of interrogating the role of transcription complexes both in HDAC isoform- selective or non-selective manner, and it has excellent potential to overcome the problem of accessibility of epitopes, false negatives, and necessity for multiple expensive antibodies. Our recent studies have demonstrated that highly potent HDAC isoform-selective PRPs can be successfully used to detect their targets in cell-free and cell-based experiments and to determine identity and proximity of other components of deacetylase complexes. We hypothesize that our highly potent bi-functional photoreactive HDAC probes can be used to recognize HDACs in transcription complexes and complement the use of HDAC antibodies in ChIP assays. To address this hypothesis, we propose the following: Aim 1) We will design and identify the chemical scaffolds, photoreactive groups (PRGs), handles for attachment of an epitope containing group (ipHandle), and the secondary recognition groups for immunoprecipitation (TagI) to achieve robust sensitivity, isoform selectivity, and reproducibility in crosslinking to chromatin-associated DC complexes. Prioritize best class I HDAC selective and non-selective PRPs for the optimization and validation in ChIP experiments. Aim 2) we will evaluate and optimize different HDAC isoform- selective and non-selective PRPs that are either already available or will be developed in Aim 1 for use in ChIP assays. We will use well-defined estrogen receptor (ER) binding sites that are known to recruit HDACs as a readout. Competition experiments will be conducted using non-photoreactive HDAC inhibitors to validate the outcomes of the PRP-based ChIP analysis. Comparisons between HDAC PRPs and HDAC antibodies will be made. Specificity of HDAC-PRPs will be evaluated using siRNA approaches. Upon completion of the proposed studies, we expect to have developed a novel approach and novel tools for studying HDACs associated with chromatin DNA. Altogether, PRP-ChIP developed in this application will serve as a foundation for future studies to understand how re-programming of the epigenome is linked to the mechanisms underlying the beneficial and adverse effects of HDAC inhibitors, thereby allowing the full clinical potential of HDAC inhibitors to be uncovered. Our ultimate goal is to use the findings of this project to speed up the development of safe HDAC-based therapeutics for cancer.